a. Genetic depletion of the enzymes HAL and AMDHD1 decreased sensitivity to methotrexate, but not to a control drug, doxorubicin. Cell viability after treatment with varying concentrations of methotrexate and doxorubicin was used to calculate the EC90s. n=3, biological replicates. Raw data of survival curves can be found in Source Data_3, sheet “Survival MTX”. b. Expression levels of AMDHD1 (left) and FTCD (right) were not significantly different across methotrexate-sensitive hematopoietic cell lines compared to methotrexate-resistant cell lines. The response to methotrexate was determined in a pooled fashion using genomically-barcoded cell lines (Fig. 1a and Extended Data Fig. 1a-c). Expression levels of AMDHD1 and FTCD were measured by qPCR and normalized to the average of control genes (UBC and HPRT). p-values were calculated using the KS test. n=4 for resistant cell lines and n=6 for sensitive cell lines (biologically independent samples). Each qPCR included three technical replicates. c. Fractional labeling of glycine by [U-13C] serine is unchanged by HAL depletion in HEL and Ramos cells in vehicle-and methotrexate-treated cells. d. Uptake of [U-13C] serine is higher in methotrexate-treated control cells but not in methotrexate-treated HAL-deficient cells. e. Glycine levels are not significantly different between HAL-deficient and control cells except for HEL cells treated with methotrexate. f. Serine levels are not significantly different between HAL-deficient and control cells except for HEL cells treated with methotrexate, where HAL-deficient HEL cells have similar levels of serine as vehicle-treated cells. For c-f: Glycine and serine levels were normalized to isotopically-labeled glutamate as an internal standard. p-values were calculated for the unlabeled fraction (c, d) or to total values (e, f) by one-way ANOVA. n=3, biological replicates. Source data for Fig. 3b-d and Extended Data Fig. 4c-f can be found in the file Source Data_3, sheet “metabolite profiling”.